Regulation of intracellular pH (pHi) is an important homeostatic function of cells. There are three major pHi regulatory mechanisms: the HCO3-/Cl- exchanger (AE), which alleviates alkalosis, and the Na+/H+ exchanger (NHE) and Na+,HCO3- /Cl- exchanger (NDBCE), both of which counteract acidosis. NHE activity, which is high at the germinal vesicle stage of oocyte, is inhibited during meiotic maturation, while this inhibition is abolished when the oocyte reaches the pronuclear (PN) stage of the zygote. On the other hand, we have previously found that NDBCE performs complementary regulation against acidosis during meiotic maturation. Additionally, we found that AE activity, which is a defense mechanism against alkalosis, gradually decreases during preimplantation period of embryonic development. Considering that NHE activity is inhibited during meiotic maturation and AE activity gradually decreases during embryonic development stages, we investigated whether NHE and NDBCE activities, both of which act against acidosis, functionally change from the PN zygote to the blastocyst stage of the embryo and identified these pH -regulating proteins at the molecular level in mice of the Balb/c strain. PN zygotes, two -cell (2-c), four -cell (4-c), morula and blastocyst stage embryos were obtained from 5 -8week -old, sexually mature female Balb/c mice by using the classical superovulation procedure. pHi was recorded by using the microspectrofluorometric technique on zygotes and embryos simultaneously loaded with the pHsensitive fluorophore, 2 ',7 '-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The activities of NHE and NDBCE were determined from the recovery curve of induced -acidosis in bicarbonate -free and bicarbonatecontaining media, respectively. Specific inhibitors such as cariporide (1 mu M), S3226 (1 and 10 mu M), EIPA (1, 5, and 25 mu M), and amiloride (1 mM) were used to functionally identify NHE isoforms, and the nonspecific inhibitor 4,4 '-diisocyanatostilbene-2,2 ' disulphonic acid, disodium salt (DIDS) was used to confirm NDBCE activity. The isoforms of the pHi-regulatory proteins were also identified by molecular biology using real-time PCR. We found that NHE activity was high at all embryonic stages, and differences between stages were not significant. Functional and molecular findings indicated that isoforms of NHE 1 and 5 are present in the blastocyst, whereas isoforms of NHE 1, 3, and 4 are functional at earlier embryonic stages. Although the contribution of NDBCE activity to recovery from induced -acidosis was detected at all embryonic stages, it was significant only in the PN zygote and the 2-c embryo. This finding was confirmed by molecular analysis, which detected the expression of SLC4A8 encoding NDBCE at all embryonic stages. In conclusion, NHE is an active and important defense mechanism against acidosis and is encoded by at least two protein isoforms in all stages of the Balb/c strain of mice. NDBCE has a supportive function in all embryonic stages, especially in the PN zygote and the 2-c embryo. Preimplantation stage embryos have effective mechanisms to defend against acidosis in response to their metabolic end products (increased acid load) and the acidic environment in utero.
